1. Field of the Invention
This invention relates to bone growth stimulation. More particularly, the invention relates to the use of dextran beads having a controlled pore size to stimulate bone and tissue growth.
2. Description of the Prior Art
In recent years, substantial research work on induced bone growth or osteogenesis has been conducted due to its clinical significance. Among all the different efforts, two separate but related approaches have brought most attention either because of their success in solving orthopedic problems or because of their considerable interest in the biology and applied science of osteoinduction. The first consists of clinical investigations of electric effect on inducing new bone formation. The second consists of biochemical investigations into bone growth, coupling, osteogenesis factors and a bone morphogenetic protein fraction.
The first approach, which has been documented at least a century ago, is to apply an electric field to stimulate and regulate osteogenesis. During the last forty years, more reports have shown that the cathode stimulates osteoinductive activity in both animal tests and clinical cases. Several studies have revealed that certain materials, such as neurones, myoblasts, neutral crest cells, epithelial cells, fibroblasts and osteoblasts migrate towards the cathode in the electric field. It could be one or a group of these materials, or some other unidentified materials, that play an important role in the process of bone regeneration.
The second approach, which started at a later time but has gained considerable attention recently, concentrates on identifying and isolating osteoinduction factors. Bone morphogenetic protein and human skeletal growth factor are the two osteoinductive proteins which have been isolated and characterized. Studies have shown that implantation of these proteins foster new bone formation.
More recently, researchers have applied charged dextran beads to enhance new bone formation. The unique characters of the dextran charged beads, their large porosity, large surface area, different charged groups and their affinity to different proteins have given some promising results. These results were reported at the 24th Annual Meeting of the Orthopaedic Research Society between Feb. 1 and 4, 1988 and published in the article "Charged Beads: Generation of Bone and Giant Cells" in the Journal of Bone & Mineral Research, 1988.
In the prior art study, three different types of Sephadex dextran beads made by Pharmacia, Inc. were used without any pretreatment. These beads are made from polyglucose dextran crosslinked with epichlorohydrin. The charged groups for producing the negative or positive charge are attached to glucose units in the matrix by stable ether linkages. The beads were suspended in Tyrode's salt solution (buffered at pH=7.3) and UV sterilized. The chemical and physical properties of these beads are listed in Table 1. The "fractionation range" refers to the ability of the beads to separate proteins having the stated molecular weights (MW).
TABLE 1 ______________________________________ Fractionation Bead Charge Counter Range (MW) Bead Charge Group Ion Globular Proteins ______________________________________ G-25 No Neutral No 1000-5000 DEA-A-25 Positive Weak Base Cl.sup.- &lt;30000 Diethyl- aminoethyl CM-C-25 Negative Weak Acid Na.sup.+ &lt;30000 Carboxy- methyl ______________________________________
In this study, only the negatively charged CM-C-25 beads displayed an osteoinductive effect. The study concluded that the negative electrical charge stimulated bone growth.
Use of charged beads to promote new bone formation may stem from one or both of the electric field inducing effect and the osteoinductive factors effect. Although quite a few studies have been conducted to investigate the effect of surface charge of the biomaterials on the bioactivity, very little effort has been taken in the study of the porosity effect of the biomaterials in the osteogenesis process.
Biomaterials with osteoinductive activity can be used not only to promote the healing of defective or fractured bone, but also to improve the integration of an existing implant with the surrounding tissue if the existing implant is coated with the bioactive materials. For the latter application, an interfacial bond of biomaterial with adjacent tissue will be the key issue to the success of implantation.
In order to understand the mechanism of biomaterial induced osteogenesis, a brief consideration of bone structure is essential. Bone is a specialized connective tissue comprising cells and an extracellular matrix (ECM). One type of cell, the osteoblast, is responsible for the fabrication of ECM. The ECM comprises organic and inorganic components. The organic component which weighs about 35% of the total weight is composed predominantly (95%) of Type I collagen. The remainder (5%) is a complex mixture of non-collagenous proteins and other macromolecules. Among these proteins, several of them, such as Bone Morphogenetic Protein (BMP), human Skeletal Growth Factor (hSGF) and some other growth factors, are known to increase cell replication and have important effects on differentiated cell function. However, little is understood of the precise modes of action of these macromolecules. The inorganic component of bone ECM is a complex calcium hydroxyapatite, more complex than the stoichiometric formula for hydroxyapatite, Ca.sub.10 (PO.sub.4).sub.6 (OH).sub.2, would suggest.
Due to the important role that osteoblasts and some osteoinductive factors play in the osteogenesis process, it is the hope that any biomaterials applied will have the capability either to colonize and concentrate the osteoinductive macromolecules, or to let the osteoblasts migrate towards the surface of these biomaterials. The unique properties of dextran beads have made them a good candidate for an osteoinductive material.
First, charging the beads offers an electric environment in the body when implanted, the same effect as an electrode can offer. Second, the different charged group and different porosity of the beads makes it possible to selectively bind to certain proteins with specific molecular weight and charge. Charged beads have been employed to separate proteins based on their different molecular weight and affinity to the beads. This characteristic, if the correct bead is chosen, makes it possible for the bead to bind and concentrate certain osteoinductive factors near the beads.
The actual osteogenesis induced by biomaterials is a complex and regulated process and the exact mechanism of it is, at the moment, not well understood. Several hypotheses have been proposed trying to correlate the in vivo/in-vitro results with the biomaterial properties, such as steric charge, porosity, particle size and the nature of the materials, etc. Among all these hypotheses, the explanation appears to lie more in the interaction between the biomaterials and the osteoinductive factors, which include Bone Morphogenetic Protein (BMP), human Skeletal Growth Factor (hSGF) and some other growth factors. The interaction could actually result in colonizing, concentrating and finally activating the osteoinductive factors involved. It has been known that some biological molecules, BMP in particular, are responsible for inducing the new bone formation. Another hypothesis emphasizes more the interaction of the biomaterials and bone cells, particularly osteoblasts, which are responsible for the fabrication of the ECM. It has been found in vitro that osteoblasts migrate towards different biomaterials at different rates and attach onto them with different morphologies depending on the surface charge of the biomaterials. However, the correlation of bone cell morphology and the osteoinductive activity is still not clear. It is also not understood that if the different cell morphology is the direct effect of the surface charge or if the effect is indirect. Due to the fact that the rate of cell migration towards the biomaterials is much slower than the rate of chemical or steric charge interaction between the macromolecules or other organic or inorganic chemicals in the physiological environment and the biomaterials, the surface charge of the biomaterials could be altered before the cells migrate and attach to the biomaterials.
In the prior art, the investigation has been only concentrated on the charge effect. It has indeed been found that osteoblast migratory morphology and extracellular matrix synthesis are sensitive to the charge of the biomaterial which is colonized. Very little attention has been paid to the effect of the porosity of the biomaterial used either as implants or as a coating on the metal implants. Because it has been known that osteoblasts colonizing a biomaterial are able to span pore openings on the surface of macroporous, bioactive substrates and the fact that the dimension of the osteoblasts is much bigger than that of the porosity studied in this art, the porosity of the beads investigated in this art probably would have little direct effect on the osteoblast migratory morphology. However, the indirect effect on osteoblast migratory morphology, which is caused by the fact that various macromolecules have different binding capability to the different pore size beads, is still possible. Because most osteoinductive macromolecules have the molecular weight range between 15,000 to 30,000, the porosity of the biomaterials used in the implants will have significant effect on the binding capability of these osteoinductive macromolecules.
The porosity of the dextran beads depends on the degree of cross-linking and the concentration of the charged groups attached to them. Sephadex A and C type beads are derived from the G-type beads by introducing the charged groups to the matrix. Although the numbers of A and C beads still remain the same as the G bead (A-25 and C-25 are made from G-25, A-50 and C-50 are made from G-50), the porosities of wet beads change significantly due to the increase of swelling capability by introducing the charged groups. Each Sephadex bead has a different molecular weight range over which molecules can be fractionated. Molecules with molecular weight above the upper limit of this range, the exclusion limit, are totally excluded from the gel. Table 2 gives the fractionation ranges for the different Sephadex dextran beads.
TABLE 2 ______________________________________ PROPERTIES OF SEPHADEX Fractionation Range (MW) Bed Peptides Volume Sephadex and Globular ml/g dry Type Proteins Dextrans Sephadex ______________________________________ G-25 1000-5000 100-5000 4-6 G-50 1500-30000 500-10000 9-11 G-75 3000-80000 1000-50000 12-15 G-100 4000-150000 1000-100000 15-20 A-25, C-25 &lt;30000 7-10 A-50, C-50 30000-150000 vary with pH ______________________________________
While dextran beads have been discussed, other polymers having properties similar to those shown above can be used. For example, an entire orthopedic implant can be made of such polymers (with the above properties) or an existing orthopedic implant can be coated with such polymers to form an osteoinductive surface thereon.